The present invention relates to the control of nematode pests.
There are nematode parasites of plants and animals, including humans. The plant parasites can cause significant economic losses in sub-tropical, tropical and temperate agriculture. Plant-parasitic nematodes are small (generally 100-300 .mu.m long but up to 4 mm long, and 15-35 .mu.m wide) worm-like animals which feed on root, stem or leaf tissues of living plants. Nematodes are present wherever plants are cultivated. Ectoparasitic nematodes, such as the dagger (Xiphinema and Longidorus spp.), stubby-root (Trichodorus and Paratrichodorus spp.) and spiral (Scutellonema and Helicotylenchus spp.) nematodes, live outside the plant and pierce the plant cells with their stylet in order to feed. Migratory endoparasitic nematodes, such as the lesion (Pratylenchus spp.), stem and bulb (Ditylenchus spp.) and burrowing (Radopholus spp.) nematodes, live and feed inside the plant, migrating through the plant tissues. Sedentary endoparasitic nematodes, such as the root-knot (Meloidogyne spp.), cyst (Globodera and Heterodera spp.), citrus (Tylenchulus spp.) and reniform (Rotylenchulus spp.) nematodes, live and feed inside the plant, inducing specialised fixed feeding sites called giant cells, syncytia or nurse cells in susceptible plants. Such fixed feeding sites serve as food transfer cells for the various developmental stages of the nematodes. Syncytia originate in the pericycle, endodermis or adjacent cortex.
Various methods have been used to control plant parasitic nematodes. They include quarantine measures, manipulation of planting and harvesting dates, improved fertilization and irrigation programmes that lessen plant stresses, crop rotation and fallowing, use of resistant and tolerant cultivars and rootstocks, organic soil amendments, and physical (eg solarization), biological and chemical control. Although quarantines are useful, especially when an infestation is first discovered, they are very expensive measures and usually cannot prevent the spread of nematodes. Furthermore, biological control is difficult to manage, and high quantitites and repeated additions of agents are required.
Today, control of plant-parasitic nematodes relies mainly on chemical control. Nematicides used commercially are generally either fumigants (eg halogenated aliphatic hydrocarbons and methyl isothiocyanate precursor compounds) or non-fumigants (eg organophosphates and oximecarbamates). However, the use of chemical nematicides is undesirable because these chemicals are highly toxic and therefore present a hazard to the user and to the environment
Thus, there is today a real need to have new, more effective, and safe means to control plant-parasitic nematodes.
Using the modern techniques of recombinant DNA and plant genetic engineering, genes encoding nematode control proteins may be cloned and introduced into cells of the appropriate crop plant, where their expression renders that plant inherently resistant to nematode attack. Genetic engineering overcomes the problem of reproductive barriers to genetic recombination.
WO 93/06710 (North Carolina State University) discloses an approach to imparting nematode resistance to plants which comprises transforming plants with a heterologous DNA construct consisting of a plant promoter, which is activated by a nematode attacking the plant, and a structural gene, which encodes a product which is toxic to the plant cells which form the feeding site of the nematode. Examples of products toxic to plant cells which are disclosed are nucleases, proteinases, toxins from plant pathogenic bacteria, lipases, membrane channel proteins and antibodies which bind to plant cell components. The disadvantage of this approach is that expression of the toxin gene must be restricted to the nematode feeding site in order to prevent death of plant cells in adjacent tissues. In practice this is difficult to achieve.
WO 92/04453 (The University of Leeds) discloses a method for conferring nematode resistance on plants by transforming plants with a heterologous DNA construct comprising a plant promoter, which is induced by nematode infection, and a structural gene encoding a product which is toxic to the plant cells forming the feeding site of the nematode or to the nematode itself. Examples of toxic products which are disclosed are enzymes such as DNase, RNase or a proteinase, antisense RNA, Bacillus thuringiensis proteins having anti-nematode activity, or an antibody which disrupts ingestion or digestion of food by the nematode. Such an approach has the disadvantage that it is ineffective against plant parasitic nematodes which do not induce the formation of specialised feeding sites.
WO 92/21757 (Plant Genetic Systems N.V.) discloses a method for conferring nematode resistance on plants which comprises transforming plants with two chimeric genes. The first chimeric gene comprises a nematode-induced promoter and a structural gene encoding a toxic product which kills the plant cells of the nematode feeding site or the nematode itself. The second chimeric gene comprises a nematode-repressed promoter and a structural gene encoding a product which, when expressed in cells of the plant inhibits or inactivates the toxic product of the first chimeric gene. Examples of types of gene products which kill plant cells or nematodes include nucleases, proteases, antisense DNA, B. thuringiensis toxins, collagenases, chitinases, glucanases, peroxidases, superoxide dismutases, lectins, glycosidases, antibacterial peptides, gelatinases, enzyme inhibitors or neurotoxins. Specific examples of gene products which can kill or disable nematodes are not disclosed. A disadvantage of this method is that it requires transformation of plants with two chimeric genes, each of which must be expressed only in specific tissues. In practice this is difficult to achieve.
WO 92/15690 (Nickerson Biocem Ltd) discloses proteinase inhibitors that have anti-nematode activity and therefore can be used to protect plants against nematodes, either by delivery of the proteinase inhibitor to nematodes or by transformation of plants with a gene coding for a proteinase inhibitor. Tests on potato plants transformed with a cowpea trypsin inhibitor (CpTI) gene, and in which detectable quantities of CpTI could measured, were found to have quantifiable effects on the rate of growth and sex ratio of cyst nematodes, and on egg numbers of root-knot nematodes but it was not demonstrated whether these effects were sufficient to reduce crop yield losses due to nematodes.
WO 92/15690 also describes tests on potato plants transformed with a pea lectin gene. Such transformed plants had little or no significant effect on cyst nematode establishment and maturation when compared to non-transformed plants.
Thus the known nematode control genes code for products which are either only partially effective or are non-selective and therefore their utilisation requires the use of additional genes to protect non-target plant cells.
Lectins are a heterogeneous class of (glyco) proteins grouped together based upon their ability to recognize and bind carbohydrate moieties of glycoconjugates. Chitin, the principal structural carbohydrate of insects, is a polymer of N-acetyl glucosamine (GluNAc) and various lectins with sugar binding specificities for GluNAc have been disclosed with insecticidal activity against certain agricultural pests.
EP-A-0351924 (Shell Internationale Research Maatschappij B.V.) relates to a transgenic plant comprising a lectin gene expressing a lectin within the plant foreign to the plant as found in nature. In particular, it discloses that pea lectin has been inserted into tobacco, and the transgenic plant has some degree of insect resistance. EP-A-0427529 (Pioneer Hi-Bred International, Inc) discloses that selected plant lectins have been found to be larvicidal against a number of common insect pests or agricultural crops.
Many lectins are known to be toxic to mammals and birds. For example, the lectins of Phaseolus vulgaris are poorly digested by rats and thus are able to react with intestinal cells causing disruption of the brush borders of duodenal and jejunal enterocytes. As a result, abnormal absorption of potentially harmful substances occurs, leading to severe toxic effects. There is a need, therefore, to identify lectins which are toxic to nematodes but at the same time do not exhibit toxicity to mammals or birds. These would be useful in crop protection applications without restriction on the food use of the material in which the foreign lectin is to be presented. WO 92/02139 (Agricultural Genetics Company Ltd) discloses that a group of lectins, characterised by specific mannose-binding ability, in particular derived from Amaryllidaceae and Alliaceae, are effective for the control of insect pests, but are non-toxic to mammals and birds. WO 91/06311 (Scottish Crop Research Institute) discloses that mannose-specific lectins obtained from Amaryllidaceae have anti-viral activity against RNA viruses such as Human Immunodeficiency Virus.